I have the expertise and experience necessary to carry out the proposed research and to supervise undergraduate and graduate students. As the number of protein-based drugs, most of which contain disulfide bonds, increases, so does the need for the efficient production of proteins via in vitro protein folding. I have published numerous papers in the field of in vitro protein folding, including two in JACS, and I have written three review articles. My prior work on protein folding focused on the in vitro oxidative folding of disulfide containing proteins with aromatic thiols. We demonstrated that aromatic thiols could significantly improve the in vitro folding rate and yield of lysozyme and RNase A even at high protein concentration. However, to improve the in vitro protein folding further we needed to better understand the origins of the enhancements by investigating how these reagents affected the folding pathway. The folding pathway of bovine pancreatic trypsin inhibitor (BPTI) is arguably the best characterized of any system but prior work had focused on the pathway and not on the overall folding rate. Initially, we optimized the in vitro folding of reduced BPTI using the standard reagents glutathione disulfide (GSSG) and glutathione (GSH) and to our surprise found that previously disregarded pathways were likely important for efficient folding. Herein, we propose to confirm the importance of these pathways and then demonstrate strategies that further improve the folding rate of BPTI with GSSG and GSH, days to hours. Aromatic disulfides will then be used to develop strategies that minimize folding times even further. Strategies that improve in vitro protein folding have the potential to improve the production of an ever-expanding number of protein-based drugs and to address protein misfolding diseases. I have a long and successful history of educating underrepresented students, including women, Hispanic and African American students. This commitment is represented in the composition of the undergraduate students in the research group. From 2004-2012, 24 undergraduate students were exposed to research in the Lees' group of which 54% were women, 58% were Hispanic and 8% were African American. These students have been coauthors on nine publications and gone on to pursue graduate degrees at Boston University, Duke, Michigan State, University of Mississippi etc., as well as pursuing professional degrees. In addition, during this time frame I have graduated 4 Masters students and 3 Ph.D. students and currently have 3 Ph.D. students in the laboratory. After graduation one of the graduate students obtained a job in academics (Univ. of Long Island) while the rest now work in the chemical/pharmaceutical industry. In summary, I have demonstrated the ability to perform productive research with graduate and undergraduate students and would like the opportunity to continue to do so.
Organic Chemistry and Biochemistry The use of organic chemistry to study biochemical problems such the folding of disulfide containing proteins Many extracellular proteins and almost all pharmaceutically relevant proteins contain disulfide bonds. The folding of these proteins in vitro is slow and methods of increasing the folding rate are being investigated.